Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a

ABSTRACT The continuous and rapid surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmissibility and evading neutralization is alarming, necessitating expeditious detection of the variants concerned. Here, we report the development of rapid SARS-CoV-2 variants enzymatic detection (SAVED) based on CRISPR-Cas12a targeting of previously crucial variants, including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, and currently circulating variant of concern (VOC) Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5. SAVED is inexpensive (US$3.23 per reaction) and instrument-free. SAVED results can be read out by fluorescence reader and tube visualization under UV/blue light, and it is stable for 1 h, enabling high-throughput screening and point-of-care testing. We validated SAVED performance on clinical samples with 100% specificity in all samples and 100% sensitivity for the current pandemic Omicron variant samples having a threshold cycle (CT) value of ≤34.9. We utilized chimeric CRISPR RNA (crRNA) and short crRNA (15-nucleotide [nt] to 17-nt spacer) to achieve single nucleotide polymorphism (SNP) genotyping, which is necessary for variant differentiation and is a challenge to accomplish using CRISPR-Cas12a technology. We propose a scheme that can be used for discriminating variants effortlessly and allows for modifications to incorporate newer upcoming variants as the mutation site of these variants may reappear in future variants. IMPORTANCE Rapid differentiation and detection tests that can directly identify SARS-CoV-2 variants must be developed in order to meet the demands of public health or clinical decisions. This will allow for the prompt treatment or isolation of infected people and the implementation of various quarantine measures for those exposed. We report the development of the rapid SARS-CoV-2 variants enzymatic detection (SAVED) method based on CRISPR-Cas12a that targets previously significant variants like Alpha, Beta, Gamma, Delta, Lambda, Mu, and Kappa as well as the VOC Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 that are currently circulating. SAVED uses no sophisticated instruments and is reasonably priced ($3.23 per reaction). As the mutation location of these variations may reoccur in subsequent variants, we offer a system that can be applied for variant discrimination with ease and allows for adjustments to integrate newer incoming variants.

differentiation of SARS-CoV-2 variants. Indeed, the specificity and accuracy of the present assay using blind clinical samples would be applicable to clinical settings. I'll provide you with a few comments below.
Comment-1 (Table 1): Too busy to compare the difference between mutation sites. Please modify Table 1 in a reader-friendly style.
Comment-2 (Figures 1o, 1u, S15, S21): As the authors mentioned in Legend for Figure1, the assay for T478K and N501Y was conducted using RNA templates because the use of DNA templates resulted in non-specific amplification. Why did this inappropriate reaction occur?
Comment-3: Please describe the reason why you set the fluorescence threshold as 5000 AU.
Comment-4 ( Figure 4): Some mutation sites such as T478K, E484A, and L452R showed no dose-dependency. The authors should discuss why the present assay resulted in the tendency.
Reviewer #2 (Comments for the Author): Dear author, Your work titled Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a has been reviewed by me. Congratulations for the hard work you put into it. While the text is generally acceptable, some points may be challenging for the reader. First of all, since the tables are very long, it will be difficult for the reader to follow. It becomes difficult to understand the subject, as repetitive expressions are included in the findings and discussion part. In addition, since two different molecular techniques are compared, it would be appropriate to mention the cost difference between them in the study. I would appreciate it if you would consider these issues. Good work.

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Figure1
, the assay for T478K and N501Y was conducted using RNA templates because the use of DNA templates resulted in non-specific amplification. Why did this inappropriate reaction occur?
Response: Many thanks for your comments. The primers of T478K and N501Y have mismatch(es) for the Omicron variant containing S477N+T478K and Q498R+N501Y. RT-RPA by T478K and N501Y primers using RNA samples decreased amplification compared RPA using DNA samples. The fluorescence signal between T478K and S477N+T478K, N501Y and Q498R+N501Y already showed obvious difference by RPA following CRISPR-Cas12a detection, with the help of reverse transcription, the signal of S477N+T478K and Q498R+N501Y lower than the threshold, which enables these mutation sites differentiation. Kindly check lines 508-512 of the marked-up manuscript or lines 399-403 of the revised manuscript.
Comment-3: Please describe the reason why you set the fluorescence threshold as 5000 AU.
Response: We used 5000 AU as the threshold and validated against spectrophotometry because below 5000 AU the signal was not visible under the UV light.    First of all, since the tables are very long, it will be difficult for the reader to follow.
Response: Thank you for your comments. We have replaced the Table 1 with Figure 1, which is much convenient for readers to follow. For the original Table 2 and Table 3, now they are Table 1 and Table 2 in the marked-up manuscript. As so many mutation sites need to be mentioned, it will lose important information if shortened.
It becomes difficult to understand the subject, as repetitive expressions are included in the findings and discussion part.

Response:
We have deleted the respective repetitive expressions in following lines: Lines 347-348: "The result of 30 min incubation was the same as 1.5 h, thus, here only shows 30 min incubation." Lines 379-382: "we used 69 clinical samples and 10 no template controls (NTC); The samples included Wild type (n=5), Alpha (n=10), Beta (n=10), Delta (n=20), Omicron BA.1 (n=5), and BA.2 (n=19) variants of SARS-CoV-2." Lines 387-389: "The sequences, variant types, and Ct value of clinical samples were unknown to the researcher until the completion of analyzing SAVED CRISPR-Cas12a-based detection data to minimize interpretation bias." Lines 453-457: "We opted for the four strategies to optimize crRNAs for detection. In this study, all selected crRNAs targeting SNP belong to chimeric or short crRNAs strategies (Figs. 1-2 and Table 1). Mutation sites with deletions or insertions can be differentiated by regular length spacer, otherwise, at least 18-nt can be used as many mismatches were present (Fig. 1d,e,f,h,i)." Lines 463-465: "As we reported previously, when we used 24-nt spacer, 20-nt spacer, and chimeric crRNAs, the specificity of chimeric crRNA was the strongest, whereas the signal was the weakest (Figs. S11-17,21) [32])." In addition, since two different molecular techniques are compared, it would be appropriate to mention the cost difference between them in the study.

Response:
We added the cost difference between our platform and RT-qPCR in Table  S3 and lines 429-430 of the marked-up manuscript or lines 320-321 of the revised manuscript.
I would appreciate it if you would consider these issues. Good work.
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Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
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